US5312922A - Europium and terbium chelators for time-resolved fluorometric assays - Google Patents

Europium and terbium chelators for time-resolved fluorometric assays Download PDF

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US5312922A
US5312922A US07/863,746 US86374692A US5312922A US 5312922 A US5312922 A US 5312922A US 86374692 A US86374692 A US 86374692A US 5312922 A US5312922 A US 5312922A
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Eleftherios P. Diamandis
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    • C12Q2334/20—Coumarin derivatives
    • C12Q2334/22—4-Methylumbelliferyl, i.e. beta-methylumbelliferone, 4MU

Definitions

  • This invention relates to florescent lanthanide metal chelates.
  • Fluorescent lanthanide metal chelates and in particular those for europium and terbium chelates have significant commercial application because of their use as labels in highly sensitive time-resolved fluorometric immunoassays (1-4).
  • the first commercially available time-resolved fluorescence immunoassay system, DelfiaTM (available by Pharmacia-LKB, Sweden) uses Eu 3+ as immunological label (5,6).
  • a second-generation time-resolved fluorometric immunoassay system uses the europium chelator 4,7-bis (chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) as immunological label (7.8).
  • BCPDA europium chelator 4,7-bis (chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid
  • Tb 3+ and its chelates have been used as immunological and nucleic acid labels (9,10).
  • Tb 3+ is inferior to Eu 3+ in terms of detectability.
  • Eu 3+ and Tb 3+ can be used simultaneously for dual analyte assays (11).
  • time-resolved fluorometric immunoassays it is desirable to use a chelate-label which can be detected down to the subpicomolar range (5).
  • multiple labelling strategies can be used in order to achieve subplcomolar analyte sensitivity (1 and 4-16).
  • the primary immunological label is alkaline phosphatase (ALP); its acid (FSAP).
  • ALP alkaline phosphatase
  • FSAP nonhydrolysed ester
  • FSA hydrolysed ester
  • FSA When FSA is added to an aqueous alkaline solution containing Tb 3+ -EDTA, a mixed complex is formed which emits long-lived fluorescence characteristic of Tb 3+ .
  • FSA is an appropriate ligand for energy transfer to the metal-ion.
  • An intact hydroxyl group on the FSA molecule is essential for these highly fluorescent mixed complexes to be formed.
  • FSAP does not form any fluorescent complexes with Tb 3+ -EDTA.
  • ALP activity can be monitored with use of FSAP as substrate, by measuring the released FSA after adding an alkaline solution of Tb 3+ -EDTA (17). This system has been used for the highly sensitive and rapid quantification of alpha-fetoprotein and thyrotropin in serum (18,19).
  • fluorescent lanthanide metal chelates are selected from compounds of the following formulas I through XXXIII: ##STR1##
  • the selected esters of chelates of formulas I through XXXIII are enzymatically treated to render the chelates either fluorescent or non-fluorescent when combined with a selected lanthanide metal.
  • FIG. 1 Logarithmic plots of fluorescence vs concentration of candidate fluorogenic lanthanide chelator. In all cases A to E but D, the fluorescence of each Eu3+ chelate was higher or equal (C) to the corresponding fluorescence of the Tb3+ chelate; the Eu3+-chelate fluorescence curve in plots A to E is thus on top of the Tb3+-chelate fluorescence curve, except in case D.
  • the chelators represented are XI (A, the third curve representing the lowest fluorescence is due to the Tb3+-EDTA-fluorosalicylate complex as described in (17); XIII (B); IX (C); XXIX (D); XXXIII (E); II (F, with Eu3+); XIV (G, with Eu3+); XIX (H, with Eu3+); XXXI (I, lower curve with Eu3+); XXXII (I, upper curve with Eu3+).
  • FIG. 2 Calibration curves for the proposed TSH assay (A). Fluorescence vs TSH concentration on linear axes (B). A percentage plot, where Bo is the fluorescence of the zero standard and B the fluorescence of all other standards. The concentration of the substrate used was (in mol/L from bottom to top): 5 ⁇ 10 -6 ; 1 ⁇ 10 -5 ; 2 ⁇ 10 -5 ; 5 ⁇ 10 -5 .
  • FIG. 3 Calibration curves for the proposed thyroxine assay (A). Double linear plots. (B) Double logarithmic plots. The substrate concentration and antibody dilutions used were: 5 ⁇ 10 -6 M and 10-fold (1); 5 ⁇ 10 -6 M and 20-fold (2); 2 ⁇ 10 -5 M and 10-fold (3); 2 ⁇ 10 -5 M and 20-fold (4).
  • FIG. 4 Correlation between serum thyroxine results obtained with a radioimmunoassay (RIA) and the proposed assay method (TR-FIA).
  • RIA radioimmunoassay
  • TR-FIA proposed assay method
  • the molecules are identified as follows in formulas I through XXXIII: ##STR2##
  • the registry numbers for these compounds is as follows: I,3-hydroxyflavone, 577-85-5; II, 4-hydroxyquinoline-2-carboxylic acid, 492-27-3; III, 2-hydroxynicotinic acid, 609-71-2; IV, 1-hydroxy-2-naphthoic acid, 86-48-6; V, 5-fluorosalicyclic acid, 345-16-4; VI, 6-hydroxynicotinic acid, 5006-66-6; VII, 2-hydroxy-6-methylpyridine-3-carboxylic acid, 38116-61-9; VIII, 3-hydroxy-2-naphthoic acid, 92-70-6; IX, 3-hydroxypicolinic acid, 874-24-8; X, 2-hydroxy-5-nitrobenzoic acid, 96-97-9; XI, 4-hydroxy-7-trifluromethyl-3-quinoline carboxylic acid, 574-92-5; XII, 3-hydroxy-4,7
  • Molecules IX, XI, XIII, XXIX and XXIII of the above formulae are good fluorogenic chelators for both Eu 3+ and Tb 3+ and molecules IX, XI, XIII, XIV, XXIX and XXXII of the above formulae are good fluorogenic chelators for Eu 3+ only. Under optimised measuring conditions of pH and presence or absence of EDTA, these chelators can be detected at levels ⁇ 10 -8 M. Thus, these substrates demonstrate utility in highly sensitive enzymatically amplified time-resolved fluorometric immunoassays.
  • the candidate organic chelators tested are shown in FIG. 1. All of them were obtained from Aldrich Chemical Co., Milwaukee, Wis. 53233. Calf intestine alkaline phosphatase (ALP) was obtained from Boehringer Mannheim Canada, Montreal PQ. Alkaline phosphatase-labelled streptavidin (SA-ALP) was obtained from Zymed Laboratories Inc., San Fransisco, Calif. 94080, as a 0.75 mg/mL solution. White, opaque 12-well polystyrene microtiter strips coated with monoclonal anti-thyrotropin antibody (for the TSH assay) or a thyroxine-globulin conjugate (for the T4 assay) were obtained from CyberFluor.
  • biotinylated detection antibodies, standards and buffers needed for the immunoassays were also from CyberFluor.
  • Europium (III) chloride hexahydrate and terbium (III) chloride hexahydrate were from Aldrich. All other chemicals were from Sigma Chemical Co., St. Louis, Mo. 63178.
  • the alkaline phosphatase substrate buffer was a 0.1 mol/L Tris, pH 9.0, containing 0.1 mol/L NaCl and 1 mmol/L MgCl 2 .
  • the diluent for the biotinylated detection TSH antibody and the SA-ALP conjugate was a 6% (w/v) solution of bovine serum albumin (BSA) in a 50 mmol/L Tris buffer, pH 7.40, containing 0.5 g of sodium azide per liter.
  • the wash solution was prepared by dissolving 9 g of NaCl and 0.5 mL of polyoxyethylenesorbltan monolaurate (Tween 20) in 1 liter of distilled water.
  • All chelators were screened by mixing 100 ⁇ L of a chelator solution and 100 ⁇ L of a Eu 3+ or Tb 3+ solution, (4 ⁇ 10 -3 mol/L in water) and measuring the delayed fluorescence on the 615TM Immunoanalyzer.
  • the chelators according to preferred embodiments of the invention were further tested with Eu 3+ or Tb 3+ solutions of various pH, in the absence or presence of EDTA.
  • Calibration curves and detection limits were constructed or calculated by adding 100 ⁇ L of an aqueous Eu 3+ or Tb 3+ solution to 100 ⁇ L of a chelator solution in a 0.1 mol/L Tris buffer of pH 9.0.
  • chelator according to formula V we added 100 ⁇ L of a Tb 3+ -EDTA solution, 10 -3 mol/L in a 0.5 mol/L Tris buffer (18) of pH 12.5.
  • chelators of formula XI and XIII we added 100 ⁇ L of a 4 ⁇ 10-3 mol/L solution of Eu 3+ or Tb 3+ in a 0.1 mol/L Tris buffer, pH 11, containing 5 ⁇ 10 -3 mol/L EDTA.
  • chelators of the formulae possess a hydroxyl group which could be converted to a phosphate ester or galactoside for the purpose of using them as substrates of alkaline phosphatase or ⁇ -galactosidase as has already been reported for the chelator of formula V which is included for comparison (17-19).
  • sets of candidate chelators with only one group difference were tested i.e. chelators of formualae XIV, XX and XXXI, to demonstrate the importance of the different groups.
  • Derivatives of a particular structure i.e.
  • chelators Two chelators, XI and XIII exhibited the property of working in the presence of excess EDTA i.e. they form fluorescent complexes with Eu 3+ and Tb 3+ chelated to EDTA. This property is also seen with chelator V (previously reported). In all other cases, fluorescence of the complexes decreases dramatically in the presence of EDTA. For all the chelators, the fluorescence measured is long-lived and lanthanide-specific. Native chelator fluorescence may also exist but is not detected by the time-resolved fluorometer.
  • Fluorescence intensity of the complexes varies with pH as shown in Table 1.
  • solutions of Eu 3+ and Tb 3+ in buffers of alkaline pH deteriorate quickly due to hydroxide precipitation.
  • complexes are preferably formed with the addition of an aqueous solution of Eu 3+ or Tb 3+ (pH ⁇ 6.5) which is stable for long periods of time at room temperature.
  • the Eu 3+ and Tb 3+ solutions were made in the presence of excess EDTA at pH 12.5 (for formula V) or 11, respectively. These solutions were also very stable when stored at room temperature.
  • the fluorescence intensity of the complexes was highest when the Eu 3+ and Tb 3+ concentration in the final solution was around 2 ⁇ 10 -3 mol/L. Lower concentrations resulted in lower fluorescence signals.
  • Fluorescence intensity remained stable ( ⁇ 10%) for at least 3 h in the case of chelators IX, XI, XIII, XIV, XXIX and XXXII and Eu 3+ and/or Tb 3+ .
  • Fluorescence intensity decreased with time (change ⁇ 10% per hour) in the case of chelators II, XIX, XXXI and XXXIII. This decrease was also noticed for chelator V and previously reported (18).
  • the modification of the structures of these chelators i.e. tranformation into phosphate esters or galactosides can provide desired spectroscopic changes and/or chelation changes and/or energy transfer changes, the net result being an increase or loss of fluorescence in the presence of the lanthanides depending on the desired effect.
  • substitution of various entities of the compounds of formulae I to XXIII may be done to enhance performance. Such substitutions may be with existing radicals or at other positions about the ringed structure. Such substition may be in regard to other halogen groups, other alkyl groups and other alkylene groups and the like.
  • Another interesting application of the invention is to have a pair of compounds A and B one of which, A, is an enzyme substrate that forms fluorescent complexes with Eu 3+ or Tb 3+ .
  • A is an enzyme substrate that forms fluorescent complexes with Eu 3+ or Tb 3+ .
  • XXXIII is a non-fluorescent ALP substrate; when ALP cleaves-off phosphate, the product, XXVI, exhibits blue, short-lived fluorescence.
  • the pair XXXIII, XXVI and Eu 3+ were used to develop two model time-resolved fluorometric immunoassays for TSH and T4 in serum.
  • the design of the TSH assay is of the non-competitive heterogeneous immunoassay format where an immunocomplex of the type solid-phase-coating antibody-TSH-detection antibody-biotin-streptavidin-alkaline phosphatase, is formed in polystyrene microtiter wells.
  • the activity of ALP can then be measured in a number of different conventional ways. In this work, we detected ALP by adding the substrate XXXIII for 30 min, followed by addition of a 4 ⁇ 1 0 -3 mol/L Eu 3+ solution to form the fluorescent complex with the unreacted substrate. The calibration curves obtained are shown in FIG. 2. The sensitivity and range of assay can be adjusted by adjusting the concentration of the substrate added.
  • T4 a competitive assay for T4 was designed as follows: Polystyrene microtiter wells were coated with a T4-protein conjugate as described previously (20). In the assay, T4 in the sample competes with immobilized T4 for binding to a biotinylated anti-T4 monclonal antibody. After washing out excess reagents, we added a SA-ALP conjugate to form the immunocomplex: solid-phase-T4-antibodybiotin-SA-ALP. The activity of ALP can then be quantified with use of the substrate, XXXIII.
  • the amount of signal generated by ALP is inversely related to the analyte concentration and the calibration curves have a shape similar to those of FIG. 2.
  • Calibration curves obtained with the system are shown in FIG. 3. Sensitivity can be adjusted by changing the antibody dilution, the substrate concentration and the substrate incubation time. Under optimised conditions, 30 clinical samples have been analyzed by the method of this invention and by a conventional radioimmunoassay procedure. The comparison is shown in FIG. 4. A good correlation exists between the two methods.
  • Enzymatically amplified time-resolved fluorescence immunoassays exhibit excellent sensitivity and can be used for the routine manual or automated assay of many analytes in biological fluids.
  • the first system reported is based on alkaline phosphatase and the substrate 5-fluorosalicyl phosphate (FSAP) in combination with Tb 3+ (17-19).
  • FSAP 5-fluorosalicyl phosphate
  • Other possible chelators can be used which offer better detectability in comparison to FSA.
  • some of these chelators form stable complexes with Eu 3+ and Tb 3+ thus being advantageous to the previously reported system in which fluorescence decays with time.

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WO1997040055A1 (en) * 1996-04-19 1997-10-30 The Dow Chemical Company Fluorescent chelates as visual tissue specific imaging agents
WO1998039654A2 (en) * 1997-03-03 1998-09-11 Akzo Nobel N.V. Diagnostic neodymium(iii), ytterbium(iii), or erbium(iii) ion-ligand complexes
US5854008A (en) * 1992-04-06 1998-12-29 Nordion International, Inc. Europium and terbium chelators for the time-resolved fluorometric assays
WO2001027699A1 (en) * 1999-10-14 2001-04-19 Meridian Chemical Technologies, Inc. Method for authentication of an item
US6329205B1 (en) 1999-08-31 2001-12-11 Molecular Probes, Inc. Detection method using luminescent europium-based protein stains
US20020187563A1 (en) * 1997-03-03 2002-12-12 Johannes Willem Hofstraat Diagnostic neodymium(iii), ytterbium(iii), or erbium(iii) ion-ligand complexes
US20030099598A1 (en) * 2001-10-22 2003-05-29 Kiefer Gary E. Tissue specific fluorescent chelates possessing long wavelength UV excitation
WO2005061725A1 (en) 2003-12-23 2005-07-07 Mount Sinai Hospital Methods for detecting markers associated with endometrial disease or phase
US20060210479A1 (en) * 2004-08-10 2006-09-21 Dow Global Technologies Inc. Targeting chelants and chelates
WO2007092627A2 (en) 2006-02-09 2007-08-16 University Of South Florida Detection of cancer by elevated levels of bcl-2
WO2009097692A1 (en) 2008-02-07 2009-08-13 Siu K W Michael Biomarkers for head-and-neck cancers and precancers
WO2010040277A1 (en) 2008-10-09 2010-04-15 The University Of Hong Kong Cadherin-17 as diagnostic marker and therapeutic target for liver cancer
US20100190662A1 (en) * 2007-01-26 2010-07-29 Rebecca Sutphen Methods and materials for detection, diagnosis and management of ovarian cancer
WO2011032296A1 (en) 2009-09-21 2011-03-24 Mount Sinai Hospital Methods and compositions for the diagnosis and treatment of thyroid cancer
WO2011137513A1 (en) 2010-05-04 2011-11-10 Paul Walfish Method for the diagnosis of epithelial cancers by the detection of epicd polypeptide
WO2012021969A1 (en) 2010-08-16 2012-02-23 Mount Sinai Hospital Markers of the male urogenital tract
CN115753699A (zh) * 2022-08-27 2023-03-07 吉林大学 一种水中四环素类抗生素的可视化检测方法

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US5854008A (en) * 1992-04-06 1998-12-29 Nordion International, Inc. Europium and terbium chelators for the time-resolved fluorometric assays
US5928627A (en) * 1996-04-19 1999-07-27 The Dow Chemical Company Fluorescent chelates as visual tissue specific imaging agents
WO1997040055A1 (en) * 1996-04-19 1997-10-30 The Dow Chemical Company Fluorescent chelates as visual tissue specific imaging agents
US20020187563A1 (en) * 1997-03-03 2002-12-12 Johannes Willem Hofstraat Diagnostic neodymium(iii), ytterbium(iii), or erbium(iii) ion-ligand complexes
WO1998039654A2 (en) * 1997-03-03 1998-09-11 Akzo Nobel N.V. Diagnostic neodymium(iii), ytterbium(iii), or erbium(iii) ion-ligand complexes
WO1998039654A3 (en) * 1997-03-03 2000-01-06 Akzo Nobel Nv Diagnostic neodymium(iii), ytterbium(iii), or erbium(iii) ion-ligand complexes
US6951760B2 (en) 1997-03-03 2005-10-04 Johannes Willem Hofstraat Diagnostic neodymium(III), ytterbium(III), or erbium(III) ion-ligand complexes
US6329205B1 (en) 1999-08-31 2001-12-11 Molecular Probes, Inc. Detection method using luminescent europium-based protein stains
WO2001027699A1 (en) * 1999-10-14 2001-04-19 Meridian Chemical Technologies, Inc. Method for authentication of an item
US20030099598A1 (en) * 2001-10-22 2003-05-29 Kiefer Gary E. Tissue specific fluorescent chelates possessing long wavelength UV excitation
US6962690B2 (en) 2001-10-22 2005-11-08 Dow Global Technologies Inc. Tissue specific fluorescent chelates possessing long wavelength UV excitation
WO2005061725A1 (en) 2003-12-23 2005-07-07 Mount Sinai Hospital Methods for detecting markers associated with endometrial disease or phase
EP2251695A2 (de) 2003-12-23 2010-11-17 Mount Sinai Hospital Corporation Marqueurs associes a la maladie de l'endometre
US20060210479A1 (en) * 2004-08-10 2006-09-21 Dow Global Technologies Inc. Targeting chelants and chelates
WO2007092627A2 (en) 2006-02-09 2007-08-16 University Of South Florida Detection of cancer by elevated levels of bcl-2
US20100190662A1 (en) * 2007-01-26 2010-07-29 Rebecca Sutphen Methods and materials for detection, diagnosis and management of ovarian cancer
WO2009097692A1 (en) 2008-02-07 2009-08-13 Siu K W Michael Biomarkers for head-and-neck cancers and precancers
EP2949790A1 (de) 2008-02-07 2015-12-02 Ranju Ralhan Biomarker für kopf- und halskrebs und krebsvorstufen
WO2010040277A1 (en) 2008-10-09 2010-04-15 The University Of Hong Kong Cadherin-17 as diagnostic marker and therapeutic target for liver cancer
WO2011032296A1 (en) 2009-09-21 2011-03-24 Mount Sinai Hospital Methods and compositions for the diagnosis and treatment of thyroid cancer
WO2011137513A1 (en) 2010-05-04 2011-11-10 Paul Walfish Method for the diagnosis of epithelial cancers by the detection of epicd polypeptide
WO2012021969A1 (en) 2010-08-16 2012-02-23 Mount Sinai Hospital Markers of the male urogenital tract
CN115753699A (zh) * 2022-08-27 2023-03-07 吉林大学 一种水中四环素类抗生素的可视化检测方法
CN115753699B (zh) * 2022-08-27 2024-05-03 吉林大学 一种水中四环素类抗生素的可视化检测方法

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EP0635006A1 (de) 1995-01-25
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